It is proposed to examine the mechanism of enzyme-catalyzed and nonenzymic reactions of the coenzyme thiamin (vitamin B1). The long-term objective of the proposed research is to determine how the intrinsic binding energy of the substrate(s) and coenzyme activates catalysis by thiamin-dependent enzymes; a particular interest is the role of the intrinsic binding energy in stabilizing or avoiding such unstable carbanion "intermediates" as the C(2)-ylide and the C(alpha)-carbanion/enamine. The immediate, short-term goals are to understand how activation of catalysis is brought about by different portions of the coenzyme on a thiamin-dependent enzyme for reactions involving these carbanion "intermediates" and to determine whether contributions from binding of a neutral tricyclic form of the coenzyme (TN) should be included in the accounting of factors contributing to activation of thiamin-dependent enzyme catalysis. The research will focus on the mechanism and catalysis of enzymic aldol- type addition-elimination reactions involving the C(2)-ylide and C(alpha)- carbanion/enamine, and the extent to which the mechanisms of these addition-elimination reactions are determined by noncovalent interactions between the enzyme and coenzyme that influence the lifetime of the carbanion "intermediate". Using standard kinetic methods, isotopic probes, and an alternate coenzyme (3,4-dimethyl-5-(2-pyrophosphoethyl)thiazolium ion, DMT) in the presence and absence of an analogue of the aminopyrimidinyl moiety (AP) of thiamin (4-amino-2-methylpyrimidine, AMP), the effect of (i) removing the covalent link between the two functional domains of thiamin and (ii) removing the AP moiety of thiamin on the free energy-reaction coordinate diagrams for aldol-type addition-elimination reactions catalyzed by pyruvate decarboxylase will be determined. This information will be used to estimate the pKa of C(2)-H in PDC-bound DMT, determine whether the C(2)-ylide and C(alpha)carbanion/enamine have a significant lifetime on PDC reconstituted with DMT in the presence and absence of AMP, and evaluate the contribution of binding of the AP moiety to activation of enzyme catalysis. Second, the mechanism for binding of thiamin to E. coli periplasmic thiamin-binding protein will be examined as an especially simple model for thiamin-dependent enzymes using standard kinetic and NMR methods and isotopic probes to determine whether binding of TN is important.